Project Summary Zika virus (ZIKV) disease poses a significant threat to people living in the endemic countries as well as travelers and military personnel. Although only 20% of ZIKV infected people develop mild symptoms (rash, fever, arthralgia or conjunctivitis), a recent major concern is that ZIKV infection has been implicated in babies with microcephaly and other congenital malformations as well as in individuals with neurological disorders (Guillain- Barr syndrome, myelitis or meninggoencephalitis). Currently, an effective and safe vaccine is urgently needed. Live attenuated virus- and chimeric virus-based approaches that are successfully used for dengue vaccine are a potential approach but there are safety concerns due to our limited understanding of ZIKV pathogenesis and to the potential for immune enhancement of disease. Additionally, safety concerns associated with the YFV backbone may limit its use in immunocompromised individuals, pregnant women, children or elderly. We have developed a flavivirus vaccine platform based on genetic fusion of the major flavivirus antigen (envelope (E) consisting of domains EI, EII & III) to bacterial flagellin (a TLR5 ligand). TLR signaling triggers an innate immune cascade that enhances antigen uptake and presentation, and facilitates induction of adaptive immune response, thereby eliminating the need for an adjuvant typically used in subunit vaccines. Our flavivirus vaccine platform has successfully used two different vaccine formats. In the `split format', EIEII is fused to the N-terminus of flagellin and the EIII domain is inserted into the D3 domain. In the `N-term format', E is fused to the N-terminus of flagellin. We have demonstrated that Dengue and West Nile vaccine candidates based on these formats can be efficiently produced in baculovirus expression system and are capable of eliciting robust virus neutralizing antibodies in mice. We here propose to construct, express, purify, and evaluate candidate ZIKV vaccines for antigenicity and TLR5 activity in vitro. Subsequently, the candidate vaccines will be compared in immunogenicity studies in mice, and then the lead candidates will be evaluated for protective efficacy in ZIKV/mouse (AG129) model established at Utah State University. We will also demonstrate that the lead candidate ZIKV vaccines induce durable immune responses that are capable of neutralizing various strains and lineages of ZIKV, and protecting mice against a lethal ZIKV challenge. The positive outcome of this study will be a solid step towards the successful development of a safe and effective vaccine to prevent ZIKV-associated diseases for people living in the endemic areas as well as travelers and military personnel, including women of childbearing age.